The aim of this research program is to investigate the anti- hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easy site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones are found by expression in bacterial, yeast, or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced, providing new insights in the discovery of novel compounds. In the current fiscal year we accomplished the first insight into the collection of gene products that are expressed in the salivary glands of 3 important vectors of human disease, the tick Ixodes scapularis and the mosquitoes Aedes aegypti and Anopheles gambiae. From this type of study, several novel molecules were discovered, and the evolutionary adaptations to blood feeding are being understood. We have also discovered novel salivary enzymatic activities, novel properties of known salivary proteins, and novel anti-coagulants with unique properties.